Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded K_v7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD₉₀ behaviour. This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD₉₀ to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1. KCNQ1 suppression-replacement (SupRep) gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement). This hybrid SupRep gene therapy was packaged into adeno-associated virus serotype 9 (AAV9) and delivered in vivo via intra-aortic root injections (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms (ECGs) were assessed in adult type 1 long QT syndrome (LQT1) and wild-type (WT) rabbits. Patch-clamp and calcium transient experiments were performed on isolated ventricular cardiomyocytes (VCMs), both at baseline and under β-adrenergic stimulation with isoproterenol. In LQT1 transgenic rabbits, treatment with KCNQ1-SupRep resulted in significant shortening of the pathologically prolonged QT interval, QT index, and cellular action potential duration (APD) and Ca²⁺T towards WT levels. Furthermore, unlike sham-treated LQT1 rabbits, SupRep-treated LQT1 rabbits demonstrated a physiological behaviour under β-adrenergic stimulation.

The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, I_Ks, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between I_Ks and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and I_Ks currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of I_Ks in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of I_Ks, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated I_Ks currents was also observed in adult cardiomyocytes from I_Ks-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell–cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of I_Ks in adult cardiomyocytes.

Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4^−/− but not in trpv4^−/− ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4^+/+ but not in trpv4^−/− myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca²⁺ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K⁺ current (IK_SS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IK_SS and to investigate the underlying signaling pathways. AP and K⁺ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2′- O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IK_SS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca²⁺ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IK_SS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IK_SS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K⁺ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K⁺ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.